1. Field of the Invention
The present invention relates to a multi-layered element for quantitative analysis of immuno-reactants, such as antigens and antibodies.
2. Related Art Statement
The antigen-antibody reaction is an immuno-reaction in which an antigen reacts with the specific antibody to be combined together, and has been widely used in the clinical examinations for detecting a trace substance in a living body and for diagnosing an autoimmune disease. However, these examinations require skilled operations, and there is a demand for a simpler method for conducting a routine examination at an adequate reproducibility. It is also demanded that a small quantity of a sample can be analyzed within a short time preferably through an automated operation for a mass examination.
Japanese Patent Laid-Open Publication Nos. 200862/1982 (EP66648A) and 77356/1984 (EP101945A) disclose multi-layered elements for the simple and automatic quantitative analyses of antigens. Japanese Patent Laid-Open Publication No. 200862/1982 discloses a multi-layered element for the analysis of an antigen, which comprises a reaction layer containing the immobilized antibody for an antigen to be analyzed, and a detection layer composed of a hydrophilic polymer. When a mixture of the analyte antigen and a predetermined amount of the labelled antigen is added to the reaction layer, competitive antigen-antibody reactions take place in the reaction layer so that the antigen and the labelled antigen, which have not been combined with the immobilized antibody, migrates into the detection layer. The amount of the labelled antigen migrating into the detection layer is detected optically to determine the amount of the antigen contained in the analyte.
The multi-layered analysis element disclosed by Japanese Patent Laid-Open Publication No. 77356/1984 comprises a spreading layer containing a fluorescence-labelled antigen, a developing layer made of a porous matrix of high liquid-retention capacity, and a reaction layer containing an immobilized antibody. When the analyte antigen is added dropwise to the spreading layer, the antigen travels to the developing layer together with the fluorescence-labelled antigen contained in the spreading layer to be retained by the developing layer. If the reaction layer does not contain the immobilized antibody, the analyte antigen and the labelled antigen can migrate only in proportion to the ratio of the liquid-retention capacity of the developing layer to that of the reaction layer. However, since the reaction layer contains the immobilized antibody, the amounts of the analyte antigen and the labelled antigen migrating into the reaction layer are increased by the amounts thereof combined with the immobilized antibody. Accordingly, as the result of competitive antigen-antibody reactions of the analyte antigen and the labelled antigen, the quantity of the analyte antigen is determined as the function of the decrease in fluorescent light intensity of the reaction layer.
The aforementioned multi-layered analysis elements are epoch-making ones since the analysis operations can be simplified, and the automated operation can be provided thereby. However, since the dye or fluorescent dye label of the antigen are directly detected in these known elements, the detection sensitivities thereof are limited in principle.
In order to improve the detection sensitivity, it could be conceived by an application of enzymeimmunoassay. In such a method, an antigen (or antibody) is labelled by an enzyme and the activity of the enzyme labelling of the antigen (or antibody) which has been combined (or not combined) with its antibody (or antigen) through the competitive antigen-antibody reaction is detected. However, in such a method, the enzyme-labelled antigen (B) combined with antibody must be separated from the enzyme-labelled antigen (F) which has not been combined with the antibody (through a so-called B/F separation), or it is necessary to use an enzyme system having an activity for B which is different from that for F.
A system which does not require the B/F separation has been known by Japanese Patent Laid-Open Publication No. 2997/1980 (US 4,238,565), in which what is utilized is a phenomenon that the chemical affinity of the coenzyme with the apoenzyme is lowered by the coupling of the antibody with an antigen which has been preliminarily combined with the coenzyme. Japanese Patent Laid-Open Publication No. 209994/1983 (EP 94,777A) teaches a method wherein depression or enhancement in enzyme activity is utilized for quantitative analysis. In this known method, an enzyme-labelled antigen is used and coupled with an enzyme inhibitor or an enzyme activator. An enzyme and a specific antibody for the analyte antigen is carried by the same carrier to be immobilized thereby. The enzyme and the specific antibody may be carried by a relatively large particle at spatially separated sites, or may be carried by separated particles of the same carrier material. As the result of competitive reactions, the enzyme inhibitor or enzyme activator combined with the immobilized enzyme inhibits or activates the immobilized enzyme, so that the enzyme activity of the entire system is depressed or enhanced. By measuring the depression or enhancement factor of the enzyme activity, the analyte antigen is quantitatively analyzed.
However, the sensitivities of the known enzymeimmunoassay methods are low in principle, since the quantity of the analyte unlabelled antigen is determined by the ratio of inhibition or activation of the enzyme activity. These methods have the disadvantages that the times for the enzymatic reactions must be prolonged for improving the sensitivities.
Accordingly, there is a demand for a multi-layered analysis element in which the enzymeimminoassay is applied and in which the B/F separation can be substantially realized for carrying out the quantitative analysis in a short time and at a high sensitivity.